The YTHDC1/GLUT3/RNF183 axis forms a positive feedback loop that modulates glucose metabolism and bladder cancer progression

Aberrant glucose metabolism is a characteristic of bladder cancer. Hyperglycemia contributes to the development and progression of bladder cancer. However, the underlying mechanism by which hyperglycemia promotes the aggressiveness of cancers, especially bladder cancer, is still incompletely understood. N6-methyladenosine (m6A) modification is a kind of methylation modification occurring at the N6 position of adenosine that is important for the pathogenesis of urological tumors. Recently, it was found that the m6A reader YTHDC1 is regulated by high-glucose conditions. In our study, we revealed that YTHDC1 is not only regulated by high-glucose conditions but is also downregulated in bladder cancer tissue and associated with the prognosis of cancer. We also showed that YTHDC1 suppresses the malignant progression of and the glycolytic process in bladder cancer cells in an m6A-dependent manner and determined that this effect is partially mediated by GLUT3. Moreover, GLUT3 was found to destabilize YTHDC1 by upregulating RNF183 expression. In summary, we identified a novel YTHDC1/GLUT3/RNF183 feedback loop that regulates disease progression and glucose metabolism in bladder cancer. Collectively, this study provides new insight regarding the pathogenesis of bladder cancer under hyperglycemic conditions and might reveal ideal candidates for the development of drugs for bladder cancer.

a, The ENCORI web tool showed the binding site of YTHDC1 on SLC2A3. b, the RMBase v2.0 web tool showed the m 6 A modification site of SLC2A3. c, the m 6 A detection region on SLC2A3. d, The ENCORI web tool was used to detect the correlation between SLC2A3 and YTHDC1 in bladder cancer (BLCA), tenosynovial giant cell tumor (TGCT), sarcoma (SARC), and Lung squamous cell carcinoma (LUSC). e, the Ensembl dataset (https://grch37-archive.ensembl.org/) was used to explore the splicing isoform of SLC2A3. f, the model depicting the SLC2A3 full length (SLC2A3-001) and the splicing isoform (SLC2A3-010). g，T24 cells were transfected indicated plasmids for 24 h. Cells were harvested for RT-PCR and RT-qPCR analysis.
The two isoforms of SLC2A3 were quantified by using the Image J software and normalizing to the amount of action beta. Data presents as mean ± SEM with three replicates. Ns, not significant; ***, P < 0.001. h, T24 cells were transfected with indicated siRNAs for 48 h. Cells were collected for RT-PCR and RT-qPCR analysis.
The two isoforms of SLC2A3 were quantified by using the Image J software and normalizing to the amount of action beta. Data presents as mean ± SEM with three replicates. Ns, not significant; **, P < 0.01; ***, P < 0.001. Supplementary Fig. 4. a and b, T24 cells were transfected with the indicated shRNAs for 48 h. Cells were harvested for ECAR and OCR assay. Data presents as mean ± SEM with three replicates. *, P < 0.05; ***, P < 0.001. c and d, T24 cell cells were transfected with the indicated plasmids for 24 h. Cells were harvested for ECAR and OCR assay. Data presents as mean ± SEM with three replicates. *, P < 0.05; ***, P < 0.001. e and f, T24 cells were transfected with the indicated shRNAs for 48 h. Cells were harvested for ECAR and OCR assay. Data presents as mean ± SEM with three replicates. Ns, not significant; *, P < 0.05; ***, P < 0.001. Supplementary Fig. 5. a, T24 and 5637 cells were transfected with the indicated shRNAs for 48 h. Cells were harvested for RT-qPCR analysis. Data presents as mean ± SEM with three replicates. **, P < 0.01; ***, P < 0.001. b, T24 and 5637 cells were transfected with the indicated plasmids for 48 h. Cells were harvested for RT-qPCR analysis. Data presents as mean ± SEM with three replicates. Ns, not significant. c, T24 cells were transfected with the indicated plasmids for 48 h. Then, these cells were treated with MG132 for another 8 h and subjected to IP and western blot analysis.

Cell migration and invasion assay
For migration assay, the cell layer in 6-well plate was scratched and detached cells were removed. For each sample, at least three scratched fields were photographed immediately. The BioCoat Matrigel invasion chamber (BD Biosciences) was used to evaluate the cell invasion ability according to the protocol of the manufacturer. Cells were cultured in the insert for 24 h. Cells were fixed in methanol for 15 min and then stained with 1 mg/ml crystal violet for 20 min. At least three fields for each group were photographed after staining, and invaded cells were counted.

In vitro angiogenesis assay
Approximately 1×10 5 HUVEC cells were resuspended with tumor-conditioned supernatant in each well of a 24-well plate containing 250 μl Matrigel (BD Bioscience, USA). The supernatant of bladder cancer cells was added to HUVEC cells. HUVEC cells were cultured and monitored for tube formation up to 12 h.

Glucose consumption and lactate production assay
Bladder cancer cells (1 × 10 5 ) were seeded in 6-well plates and cultured in DMEM medium without phenol red (Invitrogen, USA). The spent medium was collected and measured by a glucose assay kit (# ab65333, Abcam, China). Glucose consumption was calculated by subtracting the difference in glucose concentrations between the spent medium and the unused medium. Lactate levels were measured using a lactate assay kit (#ab65330, Abcam, China).

Extracellular acidification rate and oxygen consumption rate
The extracellular acidification rate (ECAR) and oxygen consumption rates (OCR) of bladder cancer cells were measured by XF24 analyzer (Seahorse Bioscience). After calibration of the analyzer, sequential injection of glucose, oligomycin A and 2-DG were used to detect the extracellular acidification rate and sequential injection of oligomycin A, FCCP, antimycin A and rotenone were used to detect the oxygen consumption rate.
Bladder cancer cells transfected with indicated constructs were seed into the 96 well plate with 5 × 10 4 per well. The next day, cells were cultured in the incubator at 37 °C with CO2 free for three hours. Then, cells were subjected to measure the extracellular acidification rate (ECAR or ECR) and oxygen consumption rates (OCR) by using the Extracellular acidification assay kit (# ab197244, Abcam) and Extracellular Oxygen Consumption kit (# ab197272, Abcam) following the manufacture's instruction. The ECAR values were collected at 60 min by micro-plate reader system using excitation and emission wavelengths of 380 and 620 nm. The OCR values were collected at 60 min by micro-plate reader system using excitation and emission wavelengths of 380 and 650 nm.

GSEA for the key gene
GSEA: KIRC patients were first divided into two groups according to the median expression level of the key gene. Then, differential expression analysis was applied between the high and low expression groups. Input genes for GSEA were sorted by their logFC values. Signaling pathways activated or suppressed by the key gene were decided by the normalized enrichment score (NES) value derived from GSEA.